Hepadnaviruses, or hepatitis B viruses, are small, hepatotropic DNA viruses that replicate through an RNA intermediate (the pregenomic RNA or pgRNA) by reverse transcription. A critical early step in viral replication is the assembly of a ribonucleoprotein (RNP) complex comprised of the viral reverse transcriptase (RT) and pgRNA, which triggers both the assembly of nucleocapsids, the site of viral DNA synthesis, and the initiation of reverse transcription, which is primed by RT itself (the protein priming reaction). Through a complex multi- step reverse transcription pathway, the pgRNA is subsequently converted, within the context of the nucleocapsids, into the characteristic, relaxed circular DNA genome which can then exit the cell. The broad, long term objective of this proposal is to elucidate the molecular mechanisms of hepadnavirus replication, focusing on the virus-cell interactions critical for viral assembly and replication. The first specific aim is to identify host factors required for the interaction between RT and pgRNA and for protein priming. A combined approach based on physical association with RT, pgRNA and viral particles and biochemical fractionation and reconstitution will be used for this purpose, taking advantage of an established cell-free system that can recapitulate these early events in viral replication. The second aim is to elucidate the determinants on both RT and pgRNA responsible for specific RNA-protein interaction. Optimal conditions for RT- binding and crosslinking will be sought. The RT and contact sites in the RNP complex will then be identified through protein and RNA sequence analyses. This will, in turn, guide further mutagenesis studies to elucidate the specific requirements of RT and pgRNA for RNP formation. The third aim is to determine the requirements for individual steps of the viral reverse transcription pathway, using a recently developed synchronized viral replication system. Nucleocapsids from every stage of reverse transcription will be isolated to determine their structural changes, which accompany, and may be required for, the progression of viral DNA synthesis. Furthermore, specific host factors required for the different steps of reverse transcription will be identified by (1) carrying out viral DNA synthesis with the isolated nucleocapsids under cell-free reconstitution conditions and (2) treating virus-producing cells, at different stages of reverse transcription, with agents that target candidate host factors. The accomplishment of these goals should reveal novel cellular, as well as viral, targets for developing effective antivirals which is urgently needed for the treatment of over 300 million patients worldwide infected with the hepatitis B virus, who are at great risk of developing hepatic failure and hepatocellular carcinoma. In addition, studies on how host factors facilitate viral replication will also provide important insights into the normal cellular functions of these factors.